Investigation of Microbes in the Rhizosphere of Selected Trees for the Rhizoremediation of Hydrocarbon-Contaminated Soils

Hydrocarbon-degrading microorganisms (HDMs), associated with the rhizosphere of Conocarpus lancifolius and Ficus infectoria trees grown in bioremediated soil, were isolated under controlled laboratory conditions. The selected trees were used to phytoremediate oil-contaminated soil for three successive growing seasons. At the end of the phytoremediation experiment, 85.7% of measurable total petroleum hydrocarbon was degraded in the rhizosphere soil associated with Conocarpus lancifolius compared to 78.6% in the rhizosphere of Ficus infectoria. The detectable concentrations of some polyaromatic hydrocarbons were less than 0.02 ppm. The HDM isolation process was conducted at 35°C under aerobic conditions. The isolated HDMs were identified using 16S rRNA gene sequencing and fatty acid methyl ester analysis. Differences in the genera of the isolated HDMs and their assessed efficiency in degrading a mixture of hydrocarbon compounds between the two trees were noted.     

Investigation of Microbes in the Rhizosphere of Selected Grasses for Rhizoremediation of Hydrocarbon-Contaminated Soils

Hydrocarbon-degrading microorganisms (HDM) associated with the rhizosphere of Paspalum vaginatum and Zoysia tenuifolia grown in bioremediated soil were isolated under controlled laboratory conditions. The isolation process was conducted at 30°C and 45°C to isolate mesophilic and thermotolerant microorganisms, respectively, under aerobic conditions. The isolated HDMs were identified using 16S rRNA gene sequencing and fatty acid methyl ester (FAME) analysis. Although differences in the genera of the isolated HDMs occurred between the two grasses, Arthrobacter spp and Bacillus spp were isolated from the rhizosphere of both plants. The efficiency of the isolated microorganisms in degrading a mixture of hydrocarbon compounds (HC) was also assessed. Among the bacterial isolates, Pseudomonas boreopolis was found to be the most effective HC degrader, while the only fungal isolate, Fusarium solani, demonstrated higher degradation rates than most of the bacterial isolates. A mixture of all the microbial isolates demonstrated a high degradation percent of HC. The isolated microorganisms thus appear to work synergistically to degrade efficiently all the tested organic compounds.     

Influence of fertilizer levels on phytoremediation of crude oil-contaminated soils with the tropical pasture grass Brachiaria brizantha (hochst. ex a. rich.) stapf

Determination of fertilizer levels in phytoremediation of petroleum hydrocarbons is a complex issue, since nutrient demands of the plant and of degrading microorganisms in the rhizosphere have to be considered In the present work, three fertilizer levels were tested in a greenhouse experiment with the aim of optimizing growth of the tropical pasture grass Brachiaria brizantha and enhance microbial degradation of heavy crude oil in soil Fertilizer was applied twice in a concentration of 200, 300, and 400 mg each of N, P, and K per kg soil before and after the first sampling (14 wk). The medium fertilizer concentration resulted in best root growth and highest absolute oil dissipation (18.4%) after 22 wk The highest concentration produced best shoot growth and highest relative oil dissipation after 14 wk (10.5% less than unplanted control). In general, degradation of total oil and grease was higher in planted than in unplanted soil, but differences diminished toward the end of the experiment. Next to fertiizer quantity, its composition is an important factor to be further studied, including the form of available nitrogen (N-NO3- vs. N-NH4+). Field trials are considered indispensable for further phytoremediation studies, since greenhouse experiments produce particular water and nutrient conditions.     

Enhanced Hydrocarbons Degradation in the Rhizosphere of Mangrove Plants by a Halophilic Bacillus Subtilis Subtilis Strain

Sulaibikhat Embayment is a severely contaminated coastline in the State of Kuwait. The contaminating pollutants include hydrocarbons, heavy metals, and suspended particles. The objective of this study is to assess the ability of mangroves planted in the Sulaibikhat Embayment to enhance hydrocarbons degradation by the activities of rhizospheric hydrocarbon degrading bacteria (HDB). Accordingly, samples were collected from the rhizosphere of selected mangrove plants and from sediments in the same location but away from mangrove marshes. The samples were analyzed chemically and microbiologically before being enriched with a mixture of hydrocarbon compounds (HC) to isolate HDB.

A number of halophilic HDB were isolated from mangroves rhizosphere and the surrounding sediments such as Pseudomonas balearica, Microbacterium barkeri and Gordonia soli. On the other hand, Bacillus velezensis and Bacillus subtilis subtilis were both isolated only from mangroves rhizosphere. Among the isolated HDB, Bacillus subtilis subtilis was distinguished with its high degradation rates of the tested HC including poly aromatic hydrocarbons. According to our knowledge, this is the first Bacillus subtilis HC-degrading strain that was isolated from Kuwait Bay and from mangroves rhizosphere.     

Monitoring the Effect of Poplar Trees on Petroleum-Hydrocarbon and Chlorinated-Solvent Contaminated Ground Water

At contaminated groundwater sites, poplar trees can be used to affect ground-water levels, flow directions, and ultimately total groundwater and contaminant flux to areas downgradient of the trees. The magnitude of the hydrologic changes can be monitored using fundamental concepts of groundwater hydrology, in addition to plant physiology-based approaches, and can be viewed as being almost independent of the contaminant released. The affect of poplar trees on the fate of groundwater contaminants, however, is contaminant dependent. Some petroleum hydrocarbons or chlorinated solvents may be mineralized or transformed to innocuous compounds by rhizospheric bacteria associated with the tree roots, mineralized or transformed by plant tissues in the transpiration stream or leaves after uptake, or passively volatilized and rapidly dispersed or oxidized in the atmosphere. These processes also can be monitored using a combination of physiological- or geochemical-based field or laboratory approaches. When combined, such hydrologic and contaminant monitoring approaches can result in a more accurate assessment of the use of poplar trees to meet regulatory goals at contaminated groundwater sites, verify that these goals continue to be met in the future, and ultimately lead to a consensus on how the performance of plant-based remedial strategies (phytoremediation) is to be assessed.     

Use of Bacteria of the Genus Azotobacter for Bioremediation of Oil-Contaminated Soils

The rate of self-purification of oil-contaminated soil increases after introduction of bacteria of the genus Azotobacter. The bacteria can assimilate oil hydrocarbons as the sole source of carbon and energy, both in the presence of fixed nitrogen and during nitrogen fixation. The species Azotobacter chroococcum activates growth of hydrocarbon-oxidizing bacteria present in Devoroil.     

Use of crops for in situ phytoremediation of polluted soils following a toxic flood from a mine spill

Following a toxic flood from a mine spill that affected over 45 km² in Southern Spain, experiments were conducted in 1999 to test the feasibility of using crops for phytoremediation of the area, after the mechanical removal of the mud. Two cereals, barley and triticale, and two Brassicaspp., rapeseed and ethiopian mustard, were planted in three contaminated plots, 50 × 100 m each, and in a control plot outside the affected area. Soil and plant contents of As, Cd, Cu, Pb, Tl and Zn were measured and bioaccumulation coefficients (BC) were calculated at maturity. The four crops tested accumulated Cd and Zn in the above-ground biomass only in the plot on acid soil. Both species of Brassica accumulated Tl (average BC of 3.6 and 1.4 for rapeseed and mustard, respectively, in contaminated plots). None of the four crop plants accumulated As, Cu and Pb under the experimental conditions. Maximum plant uptake values from soil were 5.4 mg m^–2 of As, 0.54 mg m^–2 of Cd, 9.7 mg m^–2 of Cu, 7.0 mg m^–2 of Pb, 3.4 mg m^–2 of Tl, and 260 mg m^–2 of Zn. Total crop uptake gave estimates for successful phytoremediation of at least five decades, casting doubts on the feasibility of using these crops for decontamination of the area. Nevertheless, cereal grains had mineral contents below toxicity levels for livestock, therefore it might be possible to use these crops for livestock feed while reducing deep percolation and gradually removing metals from polluted soils.     

Stimulation of hybrid poplar growth in petroleum-contaminated soils through oxygen addition and soil nutrient amendments

Hybrid poplar trees (Populus deltoides x nigra DN34) were grown in a green-house using hydrocarbon-contaminated soil from a phytoremediation demonstration site in Health, Ohio. Two independent experiments investigated the effect of nutrient addition on poplar growth and the importance of oxygen addition to root development and plant growth. Biomass measurements, poplar height, and leaf color were used as indicators of plant health in the selection of a 10/5/5 NPK fertilizer applied at 1121 kg/ha (112 kg-N, 24.4 kg-P, 46.5 kg-K per ha) to enhance hybrid poplar growth at the Health site. Five passive methods of oxygen delivery were examined, including aeration tubes, gravel addition, and an Oxygen Release Compound (ORC). When ORC was placed in coffee filters above hydrocarbon-contaminated soil, a statistically significant increase of 145% was observed in poplar biomass growth, relative to unamended controls. The ORC in filters also stimulated significant increases in root density. A 15.2-cm interval of soil directly below ORC addition exhibited an increase from 2.6 +/- 1.0 mg/cm3 to 4.8 +/- 1.0 mg/cm3, showing stimulation of root growth in hydrocarbon-stained soil. The positive response of hybrid poplars to oxygen amendments suggests that overcoming oxygen limitation to plants should be considered in phytoremediation projects when soil contamination exerts a high biochemical oxygen demand, such as in former refinery sites.     

A Counter-Current Acid Leaching Process for the Remediation of Contaminated Soils from a Small-Arms Shooting Range

The objective of this research was to use a counter-current leaching process (CCLP) with leachate treatment to develop a remediation process for contaminated soils at a small-arms shooting range (SASR). The soil contaminant concentrations were 245 mg Cu kg^?1, 3,368 mg Pb kg^?1, 73 mg Sb kg^?1, and 177 mg Zn kg^?1. The CCLP includes three acid leaching steps (1M H₂SO₄ + 4M NaCl, t = 1 h, T = 20°C, soil suspension = 100 g L^?1), followed by one water rinsing step (1 h). Seven counter-current remediation cycles were completed, and the average resulting metal removals were 93.2 ± 3.5% of Cu, 91.5 ± 5.7% of Pb, 82.2 ± 10.9% of Sb, and 30.0 ± 11.4% of Zn. The metal leaching performances decreased with the number of completed cycles. Soil treated with the CCLP with leachate treatment process met the USEPA threshold criteria of 5 mg Pb L^?1 in the TCLP leachate. The CCLP allows a decrease of the water use by 32.9 m³ t^?1 and the chemicals’ consumption by approximately 2,650 kg H₂SO₄, 6,014 kg NaCl, and 1,150 kg NaOH per ton of treated soil, in comparison to standard leaching processes. This corresponds to 78%, 69%, 83%, and 67% of reduction, respectively.     

Degradation of Polycyclic Aromatic Hydrocarbons in the Rhizosphere of Festuca arundinacea and Associated Microbial Community Changes

A phytoremediation growth chamber study was conducted to evaluate the contribution of soil microbial diversity to the contaminant degradation. Target contaminant removal from soil was assessed by monitoring concentrations of polycyclic aromatic hydrocarbons (PAHs), along with changes in the bacterial community structure over a time period of 10 months in the presence of tall fescue (Festuca arundinacea). Enhanced degradation of PAHs was observed in rhizosphere soil, with a maximum reduction in pyrene at a rate 36% higher than that noted for the unvegetated control. The dissipation of < 4-ring PAHs, 4-ring PAHs, and > 4-ring PAHs in unvegetated soil was 70%, 54%, and 49% respectively, whereas a higher dissipation rate was observed in tall fescue treated soil of 78%, 68%, and 61% at the end of the study. Microbial enumeration results showed greater total bacterial numbers and PAH-degrading bacteria in rhizosphere soil when compared to unvegetated soil. The results from the terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that there was a shift in the rhizosphere bacterial community during the phytoremediation process.     

Characterization of Plant-Growth-Promoting Effects and Concurrent Promotion of Heavy Metal Accumulation in the Tissues of the Plants Grown in The Polluted Soil by Burkholderia Strain LD-11

Plant-growth-promoting (PGP) bacteria especially with the resistance to multiple heavy metals are helpful to phytoremediation. Further development of PGP bacteria is very necessary because of the extreme diversity of plants, soils, and heavy metal pollution. A Burkholderia sp. strain, numbered LD-11, was isolated, which showed resistances to multiple heavy metals and antibiotics. It can produce indole-3-acetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase and siderophores. Inoculation with the LD-11 improved germination of seeds of the investigated vegetable plants in the presence of Cu, promoted elongation of roots and hypocotyledonary axes, enhanced the dry weights of the plants grown in the soils polluted with Cu and/or Pb, and increased activity of the soil urease and the rhizobacteria diversity. Inoculation with the LD-11 significantly enhanced Cu and/or Pb accumulation especially in the roots of the plants grown in the polluted soils. Notably, LD-11 could produce siderophores in the presence of Cu. Conclusively, the PGP effects and concurrent heavy metal accumulation in the plant tissues results from combined effects of the above-mentioned multiple factors. Cu is an important element that represses production of the siderophore by the bacteria. Phytoremediation by synergistic use of the investigated plants and the bacterial strain LD-11 is a phytoextraction process.     

重金属污染环境的植物修复及其分子机制

重金属污染物的排放和扩散造成了日益严重的环境污染。如何消除环境中的重金属污染物已成为国际性难题。近年来,植物修复技术的出现和快速发展为我们展示了一条新的有效途径：即利用植物对重金属化合物的吸收、富集和转化能力去除土壤和水体中残存的重金属污染物。文章简要介绍了重金属污染物与植物修复的关系和植物修复的生理机制,重点总结了重金属污染环境的植物修复在分子生物学方面所取得的研究进展,包括有机汞裂解酶基因merB、汞离子还原酶基因merA和金属硫蛋白基因MT等的生物学功能及其在植物修复上的应用,展望了植物修复研究工作的发展方向,并针对汞污染提出了一套修复方案。     

Potential for Plant-Based Remediation of Pesticide-Contaminated Soil and Water using Nontarget Plants such as Trees,Shrubs, and Grasses

Appropriate environmental management of pesticides includes their proper application, use of filter strips and riparian buffers to contain pesticides in runoff from fields, prompt cleanup of spills, and treatment processes for wastewater associated with manufacturing and equipment usage. Plants have beneficial effects in the management of pesticide-contaminated soil and water, including direct metabolism of many pesticides, stimulation of microbial activity in the root zone, extraction of contaminated water, and reduction of infiltrating contaminated water. In this work, we review the literature on nontarget plants that can grow in pesticide-contaminated soil and water, and the fate of pesticides in filter strips, riparian buffers, and vegetated remediation environments. Past research indicates that there are significant differences in the tolerance of plants to pesticides present in soil and water, and that some plants are more effective than others in the remediation of pesticide-contaminated soil and water. Thus, there is value in the identification of tolerant plants and favorable plant-based remediation technologies for management of pesticides and contaminated sites.     

强化植物修复重金属污染土壤的策略及其机制

重金属对生态环境、农业生产、人类健康等诸多方面造成重要危害。植物修复因其具有经济有效、绿色生态等优点,已经成为土壤重金属污染修复研究领域的热点。由于植物重金属毒害、修复耗时过长等因素致使植物修复技术受限于研究阶段而不能广泛应用于实践。采用科学合理的强化措施提高植物修复的效率可能是解决该矛盾的关键之一。讨论了根瘤菌、丛枝菌根真菌、溶磷微生物和内生真菌构建的微生物-植物共生系统在强化植物修复过程中的具体应用;概述了EDTA、EDDS等螯合剂在改变土壤中重金属可溶态,促进重金属从土壤向植株转运的重要作用;介绍了植物中编码金属转运蛋白、金属硫蛋白、植物螯合肽等与重金属转运和代谢相关的基因在植物修复领域的实际应用;归纳了上述强化策略主要机制为微生物促进植物生长、缓解重金属植物毒性以及提高了土壤中重金属生物利用度,从而促进重金属在富集植物中积累和植物生物量的增加;最后总结并展望了植物修复强化技术在今后研究的重点及存在的问题。综述植物修复技术采用的主要强化策略及其机制,旨在为利用植物修复技术治理土壤重金属污染提供重要参考。     

Rhizoremediation of Diesel-Contaminated Soil with Two Rapeseed Varieties and Petroleum degraders Reveals Different Responses of the Plant Defense Mechanisms

Plant-assisted bioremediation (rhizoremediation) stands out as a potential tool to inactivate or completely remove xenobiotics from the polluted environment. Therefore, it is of key importance to find an adequate combination of plant species and microorganisms that together enhance the clean-up process. To understand the response of plants upon bioaugmentation, the antioxidative and detoxification system was analyzed in high and low erucic acid rapeseed varieties (HEAR and LEAR, respectively), after 8 weeks of their treatment with petroleum degraders and 6000 mg diesel oil/kg dry soil. The oxidative stress was enhanced in LEAR being exposed to sole diesel oil, in comparison with HEAR. However, when LEAR plants were additionally inoculated with bacteria, suppression of total catalase (CAT) and ascorbate peroxidase (APX) activity were observed. Interestingly, glutathione transferase (GST) activity was found in these plants at a much higher level than in HEAR, which correlated with a more efficient diesel removal performed by LEAR in the polluted soil and upon bioaugmentation. A distinct profile of polycyclic aromatic hydrocarbons (PAH) was detected in leaves of these plants. Neither LEAR nor HEAR experienced any changes in the photosynthetic capacity upon diesel pollution and presence of petroleum degraders, which supports the usefulness of rhizoremediation with rapeseed.     

Model Trials for Phytoremediation of Hydrocarbon-Contaminated Sites by the Use of Different Plant Species

In order to further the development of plant-based remediation of sites contaminated by carbo-chemical and petro-chemical industries, the penetration of the roots of Phragmites australis in contaminated soil substrate was studied in model trials. The series of experiments contained model substrate with firm bitumen and tar. In terms of the level of root penetration, the roots and rhizomes penetrated equally through the middle of the pot and at the edges as well as coming up through the bottom. There were differences between the density of the root systems in the topsoil of the two variations, with the roots in the sample with a 3.5 -cm bitumen layer being more dense. The experiment also showed that shallow rooting plants can penetrate thick barriers and are suitable for planting in contaminated areas. In the subsoil zone, which contained many roots, a reduction of up to 85% in the MOH content was observed. In a second series of long-term pot experiments, the stimulation of hydrocarbon remediation by Phragmites australis, Alnus glutinosa, and Robinia pseudoacacia was studied. In the subsoil zone, which contained many roots, a reduction of up to 64% in the hydrocarbon content was determined. In the comparison between the cropped and noncropped treatments, the decontamination ratio was up to 40% higher in the cropped pots than in the pots without plants. For a determination of microbial activity, two enzymes (catalase, ß-glucosidase) and microbial biomass were measured. Variants with plants showed higher microbial activities than uncropped pots. By increasing “biostimulation,” pollution and also the leaching of pollutants can be reduced.     

Metal(loid)-Contaminated Soils as a Source of Culturable Heterotrophic Aerobic Bacteria for Remediation Applications

Heavy metal–contaminated soils are a serious environmental problem. Herein, the culturable heterotrophic bacterial community present on two metal(loid)-contaminated sites in the Northern Portugal was investigated. The bacterial counts ranged from 5.96 to 7.69 and 7.04 to 7.51 (log CFU g^?1 soil) in Sites 1 and 3, respectively. The bacterial population was predominantly composed of Firmicutes, Proteobacteria, and Actinobacteria on both sites. The most represented genera in Site 1 were Bacillus (41%) and Pseudomonas (27%), whereas Arthrobacter (21%) and Pseudomonas (13%) were the most represented genera in Site 3. Several bacterial isolates showed tolerance to high concentrations of metal(loid)s, suggesting that both contaminated sites are a valuable source of metal(loid)-tolerant bacteria, which may be further used in bioremediation and/or phytoremediation processes.     

Effects of Cd,Pb, Zn,Cu-Resistant Endophytic Enterobactersp. CBSB1 and RhodotorulaSp. CBSB79 on the Growth and Phytoextraction of Brassica Plants in Multimetal Contaminated Soils

To survey the effects of endophytic Enterobacter sp. CBSB1 and Rhodotorula sp. CBSB79 resistant to Cd²⁺, Pb²⁺, Zn²⁺, and Cu²⁺ on the growth and phytoextraction of Brassica, the endophytes were isolated by surface- sterilized methods and characterized. The CBSB1 significantly increased 44.2% of the dry weight of Brassica napus in the multimetal contaminated soil (P < 0.05) and showed no effect or declined the dry weight of B. alboglabra, B. campestris ssp. chinensis var. cummunis, B. campestris ssp. chinensis var. utilis cv. Youqing12, B. campestris ssp. chinensis var. utilis cv. Lvbao701 plants. The dry weights of B. napus, B. campestris ssp. chinensis var. utilis, and B. alboglabra showed a significant increase when the CBSB79 was inoculated (P < 0.05). In general, inoculation with bacteria and yeast did not greatly alter the metal concentration in plant tissues. Compared to Enterobacter sp. CBSB1, the yeast Rhodotorula sp CBSB79 showed higher potentials to improve extraction efficacy of Cd, Pb, Zn, and Cu by Brassica seedlings in the field.     

Analysis of Diesel Fuel Contamination in Soils by Near-Infrared Reflectance Spectrometry and Solid Phase Microextraction-Gas Chromatography

A feasibility study was undertaken to determine whether the rapid, nondestructive analytical technology, near-infrared reflectance spectrometry (NIRS) could be used to predict total petroleum hydrocarbon (TPH) in contaminated soil. Hydrocarbon concentrations were determined on samples of diesel-contaminated soils by the solid phase microextraction-gas chromatography (SPME-GC) method. The same samples were then scanned for near-infrared reflectance spectrometry over the wavelength range 1100 to 2498 nm. Calibrations were developed between the NIR spectral data and the reference SPME-GC chemical data using stepwise multiple linear regression. Linear regression relationships between NIR-predicted TPH concentrations and reference data had r² of 0.68 and 0.72. These results indicate that the combination of NIRS and SPME-GC shows promise as a method for rapid estimation of TPH in soil. A major hurdle in the evaluation of methodology for hydrocarbons residues in soil is the challenge posed by the weathering of such residues.     

Cadmium and copper uptake and accumulation by Sesbania rostrata seedling, a N-fixing annual plant: implications for the mechanism of heavy metal tolerance

Sesbania rostrata,an annual tropical legume,has been found to be tolerant to heavy metals,with an unknown mechanism.It is a promising candidate species for revegetation at mine tailings.In this study,sequential extractions with five buffers and strong acids were used to extract various chemical forms of cadmium and copper in S.rostrata,with or without Cd or Cu treatments,so that the mechanisms of tolerance and detoxification could be inferred.Both metals had low transition rates from roots to the aboveground of S.rostrata.The transition ratio of Cd (4.00%) was higher than that of Cu (1.46%).The proportion of NaCl extracted Cd (mostly in proteinbinding forms) increased drastically in Cd treated plants from being undetectable in untreated plants.This suggests that Cd induced biochemical processes producing proteinlike phytochelatins that served as a major mechanism for the high Cd tolerance of S.rostrata.The case for Cu was quite different,indicating that the mechanism for metal tolerance in S.rostrata is metal-specific.The proportion of water-insoluble Cu (e.g.oxalate and phosphate) in roots increased significantly with Cu treatment,which partially explains the tolerance of S.rostrata to Cu.However,how S.rostrata copes with the high biotic activity of inorganic salts of Cu,which increased in all parts of the plant under Cu stress,is a question for future studies.Sesbania rostrata is among the very few N-fixing plants tolerant to heavy metals.This study provides evidence for the detoxification mechanism of metals in Sesbania rostrata.